Aberrant norepinephrine [NE] neurotransmission in the human brain is linked to mood disorders, depression, drug addiction and neurodegenerative diseases. [unreadable][unreadable]-synuclein [[unreadable][unreadable]-Syn], a member of the synuclein family of proteins, is expressed in monoaminergic neurons, but its function in the brain is not known. We have shown that [unreadable].-Syn can modulate the function and trafficking of the NE transporter [NET], through interactions with the microtubule [MT] cytoskeleton. We show here that [unreadable][unreadable]-Syn can also regulate these NET activities. Such modulation by [unreadable][unreadable]-Syn is unique to NET. In a rat model of depression, there is overexpression of [unreadable][unreadable]-Syn, causing NET function and trafficking to be dysregulated and unresponsive to the effects of nocodazole [a MT destabilizing agent], probably due to tight binding of the [unreadable][unreadable]-Syn/NET complex to the MT cytoskeleton. Chronic treatment of these animals with desipramine [a NET blocker] reduces [unreadable][unreadable]-Syn protein expression, while increasing [unreadable].-Syn levels, permitting NET to be appropriately regulated by [unreadable].-Syn, with full restoration of nocodazole sensitivity. In postmortem brains from patients with depression, we also show that [unreadable][unreadable]-Syn is overexpressed, adding clinical relevance for a role for [unreadable][unreadable]-Syn in the genesis and maintenance of depression in humans. We hypothesize that [unreadable][unreadable]-Syn acts as a prodepressant, and that imbalances in [unreadable][unreadable]-Syn/[unreadable].-Syn expression levels is central to the genesis of depression. When overexpressed, [unreadable][unreadable]-Syn overrides the normative regulation of NET by [unreadable].-Syn. Therefore, targeting [unreadable][unreadable]-Syn expression levels may be key to controlling depression in humans. We will investigate here in detail the cellular and molecular mechanisms by which desipramine reduces [unreadable][unreadable]-Syn levels in both in vitro and in vivo models, which express either [unreadable][unreadable]-Syn alone or both [unreadable][unreadable]-Syn and NET, in the presence or absence of [unreadable].-Syn. We will also analyze the mechanisms by which other NET antidepressants decrease [unreadable][unreadable]-Syn expression in in vitro models. Finally, we will measure neurochemical and behavioral responses to NET antidepressants in [unreadable].-Syn overexpressing transgenic mice, as well as in [unreadable].-Syn knock-out mice. From these studies we will be able to assess the mechanisms by which [unreadable][unreadable]-Syn expression is regulated as well as ascertain the physiopathological relevance of sucrtain if DMI affects [unreadable][unreadable]-Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 [unreadable]g of cDNA sequences encoding [unreadable][unreadable]-Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and [unreadable][unreadable]-Syn levels normalized against -actin. In addition to Desipramine, we will also examine the effects of several antidepressants in T470 cells that are structurally similar and dissimilar to Desipramine, to determine if the response elicited by DMI is selective for this compound or a characteristic feature of antidepressants. Some of these antidepressants include: imipramine, reboxetine, as well as structurally un-related antidepressants, trazadone and amoxapine. Research Plan for Specific Aim 2: For these series of studies, we will use two species [and speciesappropriate non-transgenic controls] of transgenic mice: [unreadable].-Syn knock-out mice expressing [unreadable][unreadable]-Syn only and [unreadable].-Syn overexpressing mice. Adult mice of bothA levels and stability, as well as their relative distribution and co-localization after treatment with the sntidepressant. The effect of other antidepressants on [unreadable][unreadable]-Syn expression that are structurally similar and dissimilar to DMI will also be examined. Specific Aim 2: To dissect the individual participation of [unreadable].-Syn and [unreadable][unreadable]-Syn in transgenic mice [[unreadable].-Syn knock-out mice and [unreadable].-Syn overexpressing mice] in regulating NET function and their response to DMI. Results from these studies will help dissect the contribution of each of the synucleins on NET trafficking and cytoskeletal protein interactions, before and after treatment with desipramine. Research Plan for SA 1: Our published and preliminary results show that DMI reduces [unreadable][unreadable]-Syn levels in the depressive rat model, WKY rat, and in endogenously expressing cells, such as the T-470 breast carcinoma cells. We will examine the molecular mechanisms by which DMI causes a reduction in [unreadable][unreadable]-Syn levels by conducting studies in the T470 breast cancer cells which express only [unreadable][unreadable]-Syn, but not [unreadable].-Syn. Thus, time-course and dose-response studies will be followed by Western blots to analyze [unreadable][unreadable]-Syn levels, after treatment with specific levels of DMI for specific time periods. Co-immunoprecipitation studies will be conducted to measure interactions between [unreadable][unreadable]-Syn and microtubule binding proteins [MAPs, tau] and microtubules [tubulin]. Protein stability will be analyzed by pulse-chase labeling using [3H]methionine followed by immunoprecipitation with [unreadable][unreadable]-Syn antibodies. mRNA levels will be assessed by real-time RTPCR and mRNA stability will be analyzed after blockade of RNA transcription by actinomycin D (10 ug/ml). To ascertain if DMI affects [unreadable][unreadable]-Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 [unreadable]g of cDNA sequences encoding [unreadable][unreadable]-Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and [unreadable][unreadable]-Syn levels normaecific therapy. This grant is also relevant for the ARRA in that it will immediately create 3 new professional jobs.